Protein bodies (PBs) are subcellular organelles (or large vesicles, about 1-3 microns in diameter, surrounded by a membrane) that specialize in protein accumulation. They are naturally formed in some specific plant tissues, like seeds, and serve as principal source of amino acids for germination and seedling growth.
The storage proteins are co-translationally inserted into the lumen of the endoplasmic reticulum (ER) via a signal peptide to be packaged either in the ER or into the vacuoles (Galili et al., 1993 Trends Cell Biol. 3:437-443) and assembled into multimeric units inside these subcellular compartments, developing specific organelles called (ER)-derived protein bodies (PBs) or protein storage vacuoles (PSV) (Okita and Rogers, 1996 Annu. Rev. Plant Physiol Mol. Biol. 47:327-350; Herman and Larkins, 1999 Plant Cell 11:601-613; Sanderfoot and Raikel, 1999 Plant Cell 11:629-642).
The storage proteins dicotiledoneous plants are primarily soluble proteins such as the 7S globulin or vicilin type, 11S globulins or legumin-type proteins and are sequestered in PSVs together with other proteins (i.e., protease inhibitors, proteolytic enzymes, lectins and the like), sugars and salts.
In contrast to PSVs, PBs (1-3 microns) sequester predominantly prolamins, which are highly hydrophobic storage proteins of cereals (such as zeins of maize and gliadins of wheat), and lack of other auxiliary proteins (Herman et al., 1999 Plant Cell 11:601-613).
At present, no PBs have been found in tissues other than plant seeds, with the exception of the ER bodies. The ER bodies are small in size (0.2-0.4 micrometers) and are formed in Arabidopsis leaves only by wounding and chewing by insects but do not develop under normal conditions (Matsushima et al., 2003 Plant J. 33:493-502).
Genetic engineering approaches have been used to study plant PBs formation, storage protein assembly and targeting. It has been shown that when recombinant proteins, predominantly plant storage proteins are expressed and packaged in Arabidopsis and tobacco, plant tissues that did not contain PBs (as vegetative tissues), develop these organelles “de novo” (Bagga et al., 1997 Plant Cell 9:1683-1696 and Bagga et al., 1995 Plant Physiol. 107:13-23, and U.S. Pat. No. 5,990,384, U.S. Pat. No. 5,215,912, and U.S. Pat. No. 5,589,616; and Geli et al., 1994 Plant Cell 6:1911-1922).
Maize beta-zein when expressed in transgenic tobacco plants was correctly targeted in new formed ER-derived PBs in leaf cells (Bagga et al., 1995 Plant Physiol. 107:13-23). Maize gamma-zein and, truncated gamma-zein cDNAs expressed in Arabidopsis plants also accumulate in a novel ER-derived PBs in leaves (Geli et al., 1994 Plant Cell 6:1911-1922). Lysine-rich gamma-zeins expressed in maize endosperms (Torrent et al. 1997 Plant Mol. Biol. 34(1):139-149) accumulate in maize PBs and co-localized with endogenous zeins. Transgenic tobacco plants expressing alpha-zein gene demonstrated that alpha-zein was not able to form PBs. However, when alpha- and gamma-zein were co-expressed, the stability of alpha-zein increased and both proteins co-localized in ER-derived protein bodies (Coleman et al., 1996 Plant Cell 8:2335-2345). Formation of novel PBs has been also described in transgenic soybean transformed with methionine-rich 10 kDa delta-zein (Bagga et al., 2000 Plant Sci. 150:21-28).
Recombinant storage proteins are also assembled in PBs-like organelles about 100 to about 400 nm in diameter in a non-plant host system such as Xenopus oocytes and in yeast. Rosenberg et al., 1993 Plant Physiol 102:61-69 reported the expression of wheat gamma-gliadin in yeast. The gene expressed correctly and the protein was accumulated in ER-derived PBs. In Xenopus oocytes, Torrent et al., 1994 Planta 192:512-518 demonstrated that gamma zein also accumulates in PB-like organelles when transcripts encoding the protein were microinjected into oocytes. Hurkman et al., 1981 J. Cell Biol. 87:292-299 with alfa-zeins and Altschuler et al., 1993 Plant Cell 5:443-450 with gamma-gliadins had similar results in Xenopus oocytes. 
One of the fundamental achievements of the field of the biotechnology (genetic engineering) is the ability to genetically manipulate an organism to produce a protein for therapeutic, nutraceutical or industrial uses. Methods are provided for producing and recovering recombinant proteins from fermentation broth of bacteria, yeast, crop plants and mammalian cell cultures. Different approaches for protein expression in host cells have been described. The essential objectives of these approaches are: protein expression level, protein stability and protein recovery (Menkhaus et al., 2004 Biotechnol. Prog. 20: 1001-1014; Evangelista et al., 1998 Biotechnol. Prog. 14:607-614).
One strategy that can solve a problem with protein recovery is secretion. However, secretion involves some times poor expression levels and product instability. Another strategy is the accumulation of the recombinant protein in the most beneficial location in the cell. This strategy has been extensively used by directing recombinant proteins to the ER by engineering C-terminal extension of a tetrapeptide (HDEL/KDEL) (Conrad and Fiedler, 1998 Plant Mol. Biol. 38:101-109).
Fusion proteins containing a plant storage protein or storage protein domains fused to the heterologous protein have been an alternative approach to direct recombinant proteins to the ER (WO 2004003207). One interesting fusion strategy is the production of recombinant proteins fused to oleosins, constitutive protein of plant oil bodies. The specific characteristics of oil bodies benefit of the easy recovery of proteins using a two-phase system (van Rooijen and Moloney, 1995 Bio/Technology 13:72-77).
Heterologous proteins have been successfully expressed in plant cells (reviews Horn et al., 2004 Plant Cell Rep. 22:711-720; Twyman et al., 2003, Trends in Biotechnology 21:570-578; Ma et al., 1995, Science 268: 716-719; Richter et al., 2000 Nat. Biotechnol. 18:1167-1171), and in some, the expression of the recombinant protein has been directed to ER-derived PB or PSV (PSV). Yang et al., 2003 Planta 216:597-603, expressed human lysozyme in rice seeds using the seed-specific promoters of glutelin and globulin storage proteins. Immunocytochemistry results indicated that the recombinant protein was located in ER-PBs and accumulated with endogenous rice globulins and glutelins. The expression of glycoprotein B of the human cytomegalovirus (hCMV) in transgenic tobacco plants has been carried out using a glutelin promoter of rice. Tackaberry et al., 1999 Vaccine 17:3020-3029. Recently, Arcalis et al., 2004 Plant Physiology 136:1-10 expressed human serum albumin (HSA) with a C-terminal extension (KDEL) in rice seeds. The recombinant HSA accumulated in PSVs with the endogenous rice storage proteins.
One obstacle for the application of plants as biofactories is the need for more research regarding the downstream processing. Protein purification from plants is a difficult task due to the complexity of the plant system. Plant solids of the extract are large, dense and relative elevated (9-20 percent by weight) (see review Menkhaus et al., 2004 Biotechnol. Prog. 20:1001-1014). At present, recombinant protein purification techniques include clarification of the extracts, treatment with solvents to remove lipids and pigments and protein or peptides purification by several ion-exchange and gel-filtration chromatography columns. The existing protocols rely upon the use of specific solvents or aqueous solutions for each plant-host system and recombinant protein. There is a need in the art for efficient and general procedures for recombinant protein recovery from transformed hosts. This need is especially relevant in cases where recombinant proteins produced in plant hosts must to be isolated. The diversity of hosts and proteins and the different physical-chemical traits between them required an efficient method to concentrate and recover recombinant products.
Immunologic adjuvants are agents that enhance specific immune responses to vaccines and inocula. An immunologic adjuvant can be defined as any substance or formulation that, when incorporated into a vaccine or inoculum, acts generally or specifically to accelerate, prolong, or enhance the quality of specific immune responses to the immunogenic materials in the preparation.
The word adjuvant is derived from the Latin verb adjuvare, which means to help or aid. Adjuvant mechanisms of action include the following: (1) increasing the biological or immunologic half-life of vaccine or inoculum immunogens; (2) improving immunogen delivery to antigen (immunogen)-presenting cells (APCs), as well as antigen (immunogen) processing and presentation by the APCs; and (3) inducing the production of immunomodulatory cytokines.
Possession of biological activity that resembles an activity of a natural pathogen or other agent is particularly relevant for vaccines or inocula, which must induce a correct immune response in an immunized human or other animal to be effective. Several new vaccines and inocula are composed of synthetic, recombinant, or highly purified subunit immunogens (antigens) that are thought to be safer than whole-inactivated or live-attenuated vaccines. However, pathogen-related immunomodulatory adjuvant components that are typically associated with attenuated or killed pathogen vaccines are absent from such synthetic, recombinant, or highly purified subunit immunogens, which often results in weaker immunogenicity for such preparations.
Phagocytosis involves the entry of large particles, such us apoptotic cells or whole microbes, into another cell. The capacity of the cells to engulf large particles likely appeared as a nutritional function in unicellular organisms; however complex organisms have taken advantage of the phagocytic machinery to fulfill additional functions. For instance, the phagocytosis of immunogens undertaken by the macrophages, the B-cells or the dendritic cells represents a key process in innate and adaptive immunity. Indeed, phagocytosis and the subsequent killing of microbes in phagosomes form the basis of an organism's innate defense against intracellular pathogens. Furthermore, the degradation of pathogens in the phagosome lumen and the production of antigenic peptides, which are presented by phagocytic cells to activate specific lymphocytes, also link phagocytosis to adaptive immunity (Jutras et al., 2005 Annual Review in Cell Development Biology. 21:511-527).
The proteins present on and in engulfed particles encounter an array of degrading proteases in phagosomes. Yet, this destructive environment generates peptides that are capable of binding to MHC class II molecules. Newly formed immunogen-MHC class II complexes are delivered to the cell surface for presentation to CD4+ T cells (Boes et al., 2002 Nature 418:983-988). The activation of these cells induces the Th2 subset of cytokines such as IL-4 and IL-5 that help B cells to proliferate and differentiate, and is associated with humoral-type immune response.
A large body of evidence indicates that, in addition to the clear involvement of the MHC class II pathway in the immune response against phagocytosed pathogens, immunogens from pathogens, including mycobacteria, Salmonella, Brucella, and Leishmania, can elicit an immunogen cross-presentation. That is to say, the presentation of an engulfed immunogen by phagocytosis by the MHC class I-dependent response promotes the proliferation of CD8+ cytotoxic T cells (Ackerman et al., 2004 Nature Immunology 5(7):678-684; Kaufmann et al., 2005 Current Opinions in Immunology 17(1):79-87).
Dendritic cells play a central immunogen presentation role to induce the immune system (Blander et al., Nature Immunology 2006 10:1029-1035). Although rare, dendritic cells are the most highly specialized APC, with ability both to instigate and regulate immune reactivity (Lau et al. 2003 Gut 52:307-314). Although dendritic cells are important in presenting immunogens, particularly to initiate primary immune responses, macrophages are the APC type most prominent in inflammatory sites and specialized for clearing necrotic and apoptotic material. Macrophages can act not only as APC, but can also perform either pro- or anti-inflammatory roles, dependent on the means by which they are activated.
Considering that APCs play a central role in the induction and regulation of the adaptive immunity (humoral and cellular), the recognition and phagocytosis of the immunogen by those cells can be considered a key step in the immunization process. A wide variety of techniques based on the uptake of fluorescent particles have been developed to study phagocytosis by the macrophages (Vergne et al., 1998 Analytical Biochemistry 255:127-132).
An important aspect in veterinary vaccines is the genetic diversity of the species being considered and the requirement for generic systems that work across different species. To a large degree, this diversity limits the use of molecular targeting techniques to cell surface markers and immune modulators such as cytokines, because for many species including wildlife, only minimal knowledge of these molecules is available. Thus, adjuvants that rely on universal activation signals of the innate immune response (i.e. that are identical in different species) are to be preferred. Taking these requirements into consideration, particulate vaccine delivery systems are well suited for veterinary and wildlife vaccine strategies (Scheerlinck et al., 2004 Methods 40:118-124).
In Third World countries, cervical cancer (cc) is one of the major causes of cancer-related deaths. About 80% of women dying from this disease originate from low-budget countries where screening programs for early detection and the medical infrastructure for treatment are not available. In contrast, in the more developed world the mortality was reduced (by 70% in the US) during the last 50 years as a consequence of cytological screening programs [American Cancer Society, Cancer facts and figures 2004. Atlanta, Ga.] Treatment of cc patients by surgery, radiotherapy or chemotherapy results in a significant loss of quality of life. Even when optimal treatment is available about 40% of all cc patients die of this disease [Gatta et al., 1998 Eur J Cancer 34(14 Spec. No.):2218-2225]. Therefore, the development of an effective and save therapeutic vaccine is needed.
A necessary event for the development of premalignancies like cervical intraepithelial neoplasia (CIN) and cc is infection by hr-HPVs [Walboomers et al., 1999 J Pathol 189(1):12-19]. So far over 120 HPV types are identified [de Villiers et al., 2004 Virology 324(1):17-27], 18 of which were found to be associated with cc [Munoz et al., 2003 N Engl J Med 348(6):518-527].
HPV-16 is responsible for about 50% of the cases [Bosch et al., 1995 J Natl Cancer Inst 87(11):796-802]. Due to the fact that the oncoprotein E7 of the hr-HPVs is exclusively and consistently expressed by HPV-infected tumor cells [von Knebel Doeberitz et al., 1994 J Virol 68(5):2811-2821], that protein represents a specific target for an immune therapy directed against cc and its premalignant dysplasia. The E7 protein, however, is an oncoprotein with transforming activity that operates by interfering with the cell cycle control. The E7 alters cell growth regulation by inactivating the pRB (retinoblastoma) tumor suppressor protein [Dyson et al., 1989 Science 243(4893):934-937; Munger et al., 1992 Cancer Surv 12:197-217] and contains two metal-binding motifs (C-XX-C) [Edmonds et al., 1989 J Virol 63(6):2650-2656; Watanabe et al., 1990 J Virol 64(1):207-214].
For safety reasons a functional oncogene cannot be applied to humans. Therefore, efforts were made to inactivate the oncogenic properties of the HPV-16 E7. Some investigators have introduced point mutations into the sites of the E7-oncogene that are associated with transforming potential [Shi et al., 1999 J Virol 73(9):7877-7881; Smahel et al., 2001 Virology 281(2):231-238], whereas others have used HLA- (human leukocyte antigen) restricted singular epitopes [Doan et al., 2000 Cancer Res 60(11):2810-2815; Velders et al., 2001 J Immunol 166(9):5366-5373]. These approaches, however, can lead to an unwanted loss of a naturally occurring epitope that is potentially associated with a decrease in vaccine efficacy.
An aim of the present inventors was to supply several to all potential naturally occurring T cell epitopes, covering the broad range of MHC restriction. In consequence, prior knowledge of the patient's HLA-haplotype is not required. This is especially important in the outbred human population.
In addition, a more potent immune response may be induced, involving all occurring HLA-restriction elements in the vaccine. A “proof-of-principle” was generated in a study using an artificial HPV-16 E7 gene (HPV-16 E7SH) of the first generation [Osen et al., 2001 Vaccine 19(30):4276-4286]. It was shown in that study that an oncoprotein with a rearranged primary sequence still induces E7WT-specific CTLs in mice but is devoid of transforming properties. That study took advantage of the earlier finding that fusion with the VP22 gene of Herpes Simplex Virus Type 1 strongly enhances the CTL response in mice [Michel et al., 2002 Virology 294(1):47-59].
The HIV-1 virus is comprised of several layers of proteins and glycoproteins that surround its RNA, and its associated proteins integrase and reverse transcriptase. The RNA is encapsidated by a capsid protein (CA), p24. The capsid environment also contains other viral proteins such as integrase and reverse transcriptase. The capsid is in turn encapsidated by a layer of matrix protein (MA), p17. This matrix protein is associated with a lipid bilayer or envelope.
The great diversity among human immunodeficiency virus type 1 (HIV-1) subtypes, which are prevalent in various regions of the world, is a major impediment to the development of broad-based prophylactic HIV-1 vaccines. Thus, it may be necessary to develop vaccines that match local epidemics more closely (Morris et al., 2001). In southern Africa, subtype C infections predominate (UNAIDS, 2006), and isolates of this subtype have been selected for the development of a DNA vaccine in South Africa (Williamson et al., 2003). This candidate vaccine has been constructed and characterized (van Harmelen et al., 2003) and is scheduled to be evaluated in clinical trials shortly.
DNA vaccines encoding HIV or simian immunodeficiency virus (SIV)/simian-human immunodeficiency virus (SHIV) antigens have been studied extensively and shown to induce both humoral and cellular immune responses in animal models as well as in humans [Boyer et al., 1997 J Infect Dis. 176(6):1501-1509; Calarota et al., 1998 Lancet 351(9112):1320-1325; Estcourt et al., Immunol. Rev. 2004 199:144-155; Letvin et al., 1997 Proc Natl Acad Sci USA. 94(17):9378-9383; Yasutomi et al., 1996 J Virol. 70(1):678-681]. However, although DNA vaccines have been shown to be safe, immunization generates low and transient levels of immune responses. Various approaches to augment DNA vaccines have been tested [Barouch et al., 2000 Intervirology 43(4-6):282-287; Hemmi et al., 2003 J Immunol 170(6):3059-3064; Raviprakash et al., 2003 Virology. 315(2):345-352], including their use in heterologous prime-boost immunization regimens [Casimiro et al., 2003 J. Virol. 77(13):7663-7668; Cherpelis et al., 2001 Immunol Lett. 79(1-2):47-55; Leung et al., 2004 AIDS 18(7):991-1001; Pal et al., 2006 Virology 348(2):341-353; Robinson et al., 1999 Int J Mol. Med. 4(5):549-555; Suh et al., 2006 Vaccine 24(11):1811-1820. Epub 2005 Oct. 25].
The HIV-1 Gag gene encodes the precursor protein Pr55 Gag, which is the major protein that makes up the structure of the HIV viral particle. On maturation of the viral particle, Gag is cleaved by the viral protease into several smaller proteins that include the capsid (CA) protein p24, the matrix protein p17, as well as proteins p7 and p6.
HIV-1 Pr55gag precursor protein possesses an ability to self-assemble into non-replicating and non-infectious virus-like particles (VLPs) [Deml et al., 1997 Virology 235(1):26-39; Mergener et al., 1992 Virology 186(1):25-39; Sakuragi et al., 2002 Proc Natl Acad Sci USA 99(12):7956-7961; Wagner et al., 1994 Behring Inst Mitt (95):23-34; Wagner et al., 1996 Virology 220(1):128-140], and elicits strong humoral and cellular immune responses in animals [Deml et al., 1997 Virology 235(1):26-39; Deml et al., 2004 Methods Mol Med 94:133-157; Jaffray et al., 2004 J Gen Virol. 85(Pt 2):409-413], including non-human primates (NHPs) (Montefiori et al., 2001 J. Virol. 75(21):10200-10207; Paliard et al., 2000 AIDS Res Hum Retroviruses 16(3):273-282]. Recently, Chege et al., J Gen Virol 2008 89:2214-2227 have shown that subtype C Pr55gag VLPs can very efficiently boost baboons primed with a matched DNA vaccine.
In addition, HIV-1 Pr55gag VLPs are safe, easy to produce and have the potential of including chimeric immunogens (Doan et al., 2005; Halsey et al., 2008). Their particulate nature and size, which approximates that of HIV-1, make HIV-1 Pr55gag VLPs more likely to stimulate the immune system better than non-particulate immunogens.
As above, the p24 protein forms the outer capsid layer of the viral particle. This protein has a high density of cytotoxic T-lymphocyte (CTL) epitopes compared to other parts of the HIV proteome (Novitsky et al., J. Virol. 2002 76(20):10155-10168), which make it more effective in inducing a broad immune response when used as a vaccine candidate. It has also been shown that the risk of AIDS is greatly increased in individuals with falling titres of p24 antibodies. This suggests that high anti-p24 antibody titres might be necessary to maintain a disease-free state.
In addition, HIV-1 Pr55gag VLPs are safe, easy to produce and have the potential of including chimeric immunogens [Doan et al., 2005 Rev Med. Virol. 15(2):75-88; Halsey et al., 2008 Virus Res. 2008 133(2):259-268. Epub 2008 Mar. 10]. Their particulate nature and size, which approximates that of HIV-1, make HIV-1 Pr55gag VLPs more likely to stimulate the immune system better than non-particulate immunogens.
As above, the p24 protein forms the outer capsid layer of the viral particle. This protein has a high density of cytotoxic T-lymphocyte (CTL) epitopes compared to other parts of the HIV proteome (Novitsky et al., J. Virol. 2002 76(20):10155-10168), which make it more effective in inducing a broad immune response when used as a vaccine candidate. It has also been shown that the risk of AIDS is greatly increased in individuals with falling titres of p24 antibodies. This suggests that high anti-p24 antibody titres might be necessary to maintain a disease-free state.
The matrix protein, p17, facilitates the intra-membrane associations that are required for viral assembly and release (Dorfman et al., 1994 J Virol 68(12):8180-8187]. Protein p17 is also involved in the transport of the viral pre-integration complex into the nucleus (Burkinsky et al., 1993). Fused together with p24, this p17/p24 (p41) complex contains the highest density of CTL epitopes in the HIV-1 genome (Novitsky et al., J. Virol. 2002 76(20):10155-10168).
HIV-1 reverse transcriptase (RT) is an RNA-dependent DNA polymerase that makes DNA templates and synthesises DNA from RNA. It is essential for viral replication. HIV-1 RT is cleaved from the Pr160gag-pol polyprotein by the HIV-1 protease (PR). Several CTL epitopes against HIV-1 have been identified in RT, although they appear to be subdominant to Gag-specific epitopes [Dela Cruz et al., 2000 Int Immunol 12(9):1293-1302].
Several studies have indicated that enhanced immune responses can be achieved by heterologous prime-boost inoculation regimens. It has been shown that a HIV-1 DNA vaccine denominated pTHGagC used as a prime inoculation of mice is boosted effectively by Pr55Gag virus-like particles (VLPs) (Chege et al., J. Gen. Virol. 2008 89:2214-2227). Because p24 has the highest density of cytotoxic T-lymphocyte (CTL) epitopes compared to other parts of the HIV proteome it was thought that the particulate nature of protein bodies containing p24 may have a similar boosting effect to expand immune responses after the immune system has been primed. It has also been thought that the use of combinations of protein bodies containing different HIV-1 antigens such as p41 and RT, may broaden the immune response such as has been shown in the use of the multigene DNA vaccine “grttn” (Burgers et al., AIDS Research and Human Retroviruses 2008 24(2):195-206) that contains five AIDS genes, the gag, reverse transcriptase, tat and nef genes that are expressed as a polyprotein and a truncated env gene (gp150).